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The unregulated uptake of modified low-density lipoproteins (LDL) by macrophages leads to foam cell formation, promoting atherosclerotic plaque progression. The cholesterol efflux capacity of macrophages by the ATP-Binding Cassette transporters depends on the ATP mitochondrial production. Therefore, the mitochondrial function maintenance is crucial in limiting foam cell formation. Thus, we aimed to investigate the mechanisms involved in the mitochondrial dysfunction that may occur in cholesterol-laden macrophages. We incubated THP-1 macrophages with acetylated LDL (acLDL) to obtain cholesterol-laden cells or with mildly oxidized LDL (oxLDL) to generate cholesterol- and oxidized lipids-laden cells. Cellular cholesterol content was measured in each condition. Mitochondrial function was evaluated by measurement of several markers of energetic metabolism, oxidative phosphorylation, oxidative stress, mitochondrial biogenesis and dynamics. OxLDL-exposed macrophages exhibited a significantly reduced mitochondrial respiration and complexes I and III activities, associated to an oxidative stress state and a reduced mitochondrial DNA copy number. Meanwhile, acLDL-exposed macrophages featured an efficient oxidative phosphorylation despite the decreased activities of aconitase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Our study revealed that mitochondrial function was differently impacted according to the nature of modified LDL. Exposure to cholesterol and oxidized lipids carried by oxLDL leads to a mitochondrial dysfunction in macrophages, affecting the mitochondrial respiratory chain functional capacity, whereas the cellular cholesterol enrichment induced by acLDL exposure results in a tricarboxylic acid cycle shunt while maintaining mitochondrial energetic production, reflecting a metabolic adaptation to cholesterol intake. These new mechanistic insights are of direct relevance to the understanding of the mitochondrial dysfunction in foam cells.

Adenosine kinase (ADK) deficiency is characterized by liver disease, dysmorphic features, epilepsy and developmental delay. This defect disrupts the adenosine/AMP futile cycle and interferes with the upstream methionine cycle. We report the clinical, histological and biochemical courses of three ADK children carrying two new mutations and presenting with neonatal cholestasis and neurological disorders. One of them died of liver failure whereas the other two recovered from their liver damage. As the phenotype was consistent with a mitochondrial disorder, we studied liver mitochondrial respiratory chain activities in two patients and revealed a combined defect of several complexes. In addition, we retrospectively analyzed methionine plasma concentration, a hallmark of ADK deficiency, in a cohort of children and showed that methionine level in patients with ADK deficiency was strongly increased compared with patients with other liver diseases. ADK deficiency is a cause of neonatal or early infantile liver disease that may mimic primary mitochondrial disorders. In this context, an elevation of methionine plasma levels over twice the upper limit should not be considered as a nonspecific finding. ADK deficiency induced-liver dysfunction is most often transient, but could be life-threatening.

International audience; Atherogenic LDL particles are physicochemically and metabolically heterogeneous. Can bioactive lipid cargo differentiate LDLsubclasses, and thus potential atherogenicity? What is the effect of statin treatment? Obese hypertriglyceridemic hypercholesterolemicmales [n = 12; lipoprotein (a)

First-trimester placenta (2 pressure (12–14 GW), the activities of the antioxidant enzymes SOD1, catalase and GPX1 are increased. The redox-sensitive MAPK pathways show increased phosphorylated-p38 expression, but no variation in the phosphorylation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) during first trimester, suggesting a physiological redox adaptation, whilst ERK1/2 phosphorylation is higher after 12 GW. Nox is the major superoxide source in early pregnancy (

Resume La biotine est utilisee librement a des doses fortes (≥ 10 mg) pouvant entrainer des problemes d’interference sur les dosages immunologiques. Le laboratoire doit maitriser l’impact de cette interference. L’objectif de l’etude a ete, d’une part, d’evaluer in vitro le niveau d’interference de la biotine sur l’automate Cobas 8000 e602®, Roche Diagnostics™ sur les resultats des marqueurs cardiaques, du bilan thyroidien, des hormones gonadotropes, de la procalcitonine, des folates et de la vitamine BI2. D’autre part, d’evaluer in vivo chez des sujets sains, l’effet de la prise de biotine (5 mg, 3 fois par jour) pendant 5 jours sur les resultats du bilan thyroidien. Les analyses ont ete faites sur le Cobas e602® et sur l’automate Advia Centaur XP® (Siemens™). L’interference in vitro de la biotine consiste en une sous-estimation des resultats pour les dosages en methode sandwich et en une surestimation en cas de methode par competition. Une variation > 10 % est observee a partir d’une concentration en biotine de 20 ng/mL pour TnThs, TSH et FT4, de 30 ng/mL pour FT3 et PCT, et superieure a 50 ng/mL pour les autres parametres. L’etude in vivo montre une diminution des concentrations de TSH et une augmentation de celles de FT3 mais avec des evolutions differentes entre le Cobas® et le Centaur® suggerant un impact de la biotine dans les variations observees. Si la plupart des resultats des parametres immunologiques realises sur Cobas e602® peuvent etre affectes suite a un traitement du patient par de la biotine a des doses superieures ou egales a 5 mg, 3 fois par jour, cet effet ne sera notable que pour certains parametres, TSH, FT4, FT3, TnThs et PCT.

Background Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. Results We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. Conclusions These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

International audience; More than ever, the circulating concentration of low-density lipoprotein–cholesterol (LDL-C) has become a key criterion for the assessment of cardiovascular risk worldwide, and in turn, for clinical management of such risk. This “fait accompli” in large part reflects the recognition of the causality of LDL particles in the pathophysiology of atherosclerotic cardiovascular disease (ASCVD), and of the robust body of evidence demonstrating that efficacious and sustained lowering of LDL-C concentrations over time confers marked reduction in both risk and cardiovascular events (1,). As a consequence, many national and international guidelines for ASCVD prevention now focus on LDL-C targets as a function of the level of global risk in secondary prevention...

Troponin is a specific cardiac infarction isoform (TnIc, TnTc) and its determination is used for the diagnosis of myocardial infarction even with normal Electrocardiography. The increase of cardiac troponins occurs in a variety of clinical situations without an acute coronary syndrome (ACS), cardiologists and emergency physicians are often confronted with positive troponins that are difficult to interpret. Few data exist about the occurrence, the clinical characteristics and the predictive value in case of absence of ACS. The objective of this study is to present the main extracardiac causes responsible of the increase of TnIc. We present some clinical cases that illustrate this diagnostic problem. A troponin elevation is observed in myopericarditis, renal failure, heart failure, pulmonary embolism, septic shock, rhabdomyolysis, stroke and others where there is a myocardial damage unrelated to coronary occlusion. Many cases of false positives, which raise the possibility of analytical interferences, must be identified.

International audience; BACKGROUND:Familial apolipoprotein A-I (apoA-I) deficiency (FAID) involving low levels of both apoA-I and high-density lipoprotein (HDL) cholesterol is associated with accelerated atherosclerosis.OBJECTIVE:The objective of this study was to define distinctive patterns in the lipidome of HDL subpopulations in FAID in relationship to antiatherogenic activities.METHODS:Five HDL subfractions were isolated by ultracentrifugation from plasma of FAID Caucasian patients (n = 5) and age-matched healthy normolipidemic Caucasian controls (n = 8), and the HDL lipidome (160 molecular species of 9 classes of phospholipids and sphingolipids) was quantitatively evaluated.RESULTS:Increased concentrations of numerous molecular species of lysophosphatidylcholine (up to 12-fold), ceramides (up to 3-fold), phosphatidylserine (up to 34-fold), phosphatidic acid (up to 71-fold), and phosphatidylglycerol (up to 20-fold) were detected throughout all five HDL subpopulations as compared with their counterparts from controls, whereas concentrations of phosphatidylethanolamine species were decreased (up to 5-fold). Moderately to highly abundant, within their lipid class, species of phosphatidylcholine, sphingomyelin, phosphatidylinositol, phosphatidylethanolamine, phosphatidylserine, and ceramide featuring multiple unsaturations were primarily affected by apoA-I deficiency; their HDL content, particularly that of phosphatidylcholine (34:2), was strongly correlated with HDL function, impaired in FAID. Metabolic pathway analysis revealed that sphingolipid, glycerophospholipid, and linoleic acid metabolism was significantly affected by FAID.CONCLUSION:These data reveal that altered content of specific phospholipid and sphingolipid species is linked to deficient antiatherogenic properties of HDL in FAID.

Rationale: CSL112, human apolipoprotein A-I (apoA-I) reconstituted with phosphatidylcholine, is known to cause a dramatic rise in small high-density lipoprotein (HDL). Objective: To explore the mechanisms by which the formation of small HDL particles is induced by CSL112. Methods and Results: Infusion of CSL112 into humans caused elevation of 2 small diameter HDL fractions and 1 large diameter fraction. Ex vivo studies showed that this remodeling does not depend on lipid transfer proteins or lipases. Rather, interaction of CSL112 with purified HDL spontaneously gave rise to 3 HDL species: a large, spherical species composed of apoA-I from native HDL and CSL112; a small, disc-shaped species composed of apoA-I from CSL112, but smaller because of the loss of phospholipids; and the smallest species, lipid-poor apoA-I composed of apoA-I from HDL and CSL112. Time-course studies suggest that remodeling occurs by an initial fusion of CSL112 with HDL and subsequent fission leading to the smaller forms. Functional studies showed that ATP-binding cassette transporter 1–dependent cholesterol efflux and anti-inflammatory effects in whole blood were carried by the 2 small species with little activity in the large species. In contrast, the ability to inactivate lipid hydroperoxides in oxidized low-density lipoprotein was carried predominantly by the 2 largest species and was low in lipid-poor apoA-I. Conclusions: We have described a mechanism for the formation of small, highly functional HDL species involving spontaneous fusion of discoidal HDL with spherical HDL and subsequent fission. Similar remodeling is likely to occur during the life cycle of apoA-I in vivo.

Familial hypercholesterolemia (FH) features elevated oxidative stress and accelerated atherosclerosis driven by elevated levels of atherogenic lipoproteins relative to subnormal levels of atheroprotective high-density lipoprotein (HDL). Small, dense HDL3 potently protects low-density lipoprotein (LDL) against proinflammatory oxidative damage.To determine whether antioxidative and/or anti-inflammatory activities of HDL are defective in FH and whether such defects are corrected by LDL apheresis.Antioxidative and antiinflammatory activities of HDL were evaluated as protection of reference LDL from oxidative stress and capacity to prevent accumulation of proinflammatory oxidised lipids, respectively. Lipid surface rigidity of HDL was assessed using a fluorescent probe. HDL components were measured by analytical approaches. Systemic oxidative stress was characterized as plasma 8-isoprostanes.Pre-LDL-apheresis, FH patients (n = 10) exhibited elevated systemic oxidative stress (3.3-fold, P0.001) vs. sex- and age-matched normolipidemic controls (n = 10). Both antioxidative and antiinflammatory activity of HDL3 were impaired (up to -91%, P0.01) in FH. Sphingomyelin and saturated fatty acid contents were elevated in FH HDL3, resulting in enhanced lipid surface rigidity. The surface lipid content (phospholipids, free cholesterol) was reduced in FH (up to -15%, P0.001), whereas content of core lipids (cholesteryl esters, triglycerides) was elevated (up to +17%, P0.001). Molar apolipoprotein A-I content of HDL3 was subnormal in FH. A single LDL-apheresis session partially corrected (by up to 76%) deficient HDL antiatherogenic activities, attenuated systemic oxidative stress and partially normalised both the lipid composition and surface rigidity of HDL particles.FH features elevated oxidative stress and deficient antioxidative and anti-inflammatory activities of small, dense HDL3; such functional deficiency is intimately linked to anomalies in lipid and protein composition, which may impair the capacity of HDL to acquire and inactivate oxidized lipids.

IF 4.810; International audience; The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

Sex steroids regulate brain function in both normal and pathological states. Mitochondria are an essential target of steroids, as demonstrated by the experimental administration of 17β-estradiol or progesterone (PROG) to ovariectomized female rodents, but the influence of endogenous sex steroids remains understudied. To address this issue, mitochondrial oxidative stress, the oxidative phosphorylation system, and brain steroid levels were analyzed under 3 different experimental sets of endocrine conditions. The first set was designed to study steroid-mediated sex differences in young male and female mice, intact and after gonadectomy. The second set concerned young female mice at 3 time points of the estrous cycle in order to analyze the influence of transient variations in steroid levels. The third set involved the evaluation of the effects of a permanent decrease in gonadal steroids in aged male and female mice. Our results show that young adult females have lower oxidative stress and a higher reduced nicotinamide adenine dinucleotide (NADH)-linked respiration rate, which is related to a higher pyruvate dehydrogenase complex activity as compared with young adult males. This sex difference did not depend on phases of the estrous cycle, was suppressed by ovariectomy but not by orchidectomy, and no longer existed in aged mice. Concomitant analysis of brain steroids showed that pregnenolone and PROG brain levels were higher in females during the reproductive period than in males and decreased with aging in females. These findings suggest that the major male/female differences in brain pregnenolone and PROG levels may contribute to the sex differences observed in brain mitochondrial function.

Strong epidemiological evidence supports a causal relationship between dyslipidemia and atherosclerotic cardio-vascular disease, which remains the leading cause of death and morbidity worldwide. A lipid profile (total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides) is recommended at the initial evaluation for the assessment of the cardio-vascular risk. A commentary of the lipid profile for the clinician and mostly for the patient should be stated on the lab report. Quantifying the cardio-vascular risk using the SCORE (systematic coronary risk estimation) system, as recommended by the Haute autorite de sante (HAS), is the starting point to establish therapeutic goals and treatment strategies. It may be important to emphasize therapeutic goals in lipid reports in order to better monitor lipid profiles at appropriate intervals to assess compliance and therapeutic efficacy for patients on lipid lowering therapy.

Steroids are neuroprotective and a growing body of evidence indicates that mitochondria are a potential target of their effects. The mitochondria are the site of cellular energy synthesis, regulate oxidative stress and play a key role in cell death after brain injury and neurodegenerative diseases. After providing a summary of the literature on the general functions of mitochondria and the effects of sex steroid administrations on mitochondrial metabolism, we summarise and discuss our recent findings concerning sex differences in brain mitochondrial function under physiological and pathological conditions. To analyse the influence of endogenous sex steroids, the oxidative phosphorylation system, mitochondrial oxidative stress and brain steroid levels were compared between male and female mice, either intact or gonadectomised. The results obtained show that females have higher a mitochondrial respiration and lower oxidative stress compared to males and also that these differences were suppressed by ovariectomy but not orchidectomy. We have also shown that the decrease in brain mitochondrial respiration induced by ischaemia/reperfusion is different according to sex. In both sexes, treatment with progesterone reduced the ischaemia/reperfusion-induced mitochondrial alterations. Our findings indicate sex differences in brain mitochondrial function under physiological conditions, as well as after stroke, and identify mitochondria as a target of the neuroprotective properties of progesterone. Thus, it is necessary to investigate sex specificity in brain physiopathological mechanisms, especially when mitochondria impairment is involved.

International audience; The aim of the present study was to develop and characterize a resveratrol self-emulsifying drug delivery system (Res-SEDDS), and to compare the uptake of resveratrol by bovine aortic endothelial cells (BAECs), and the protection of these cells against hydrogen peroxide-mediated cell death, versus a control resveratrol ethanolic solution. Three Res-SEDDSs were prepared and evaluated. The in vitro self-emulsification properties of these formulations, the droplet size and the zeta potential of the nanoemulsions formed on adding them to water under mild agitation conditions were studied, together with their toxicity on BAECs. An optimal atoxic formulation (20% Miglyol((R)) 812, 70% Montanox((R)) 80, 10% ethanol 96% v/v) was selected and further studied. Pre-incubation of BAECs for 180 min with 25 mu M resveratrol in the nanoemulsion obtained from the selected SEDDS significantly increased the membrane and intracellular concentrations of resveratrol (for example, 0.076 +/- 0.015 vs. ethanolic solution 0.041 +/- 0.016 nmol/mg of protein after 60 min incubation, p < 0.05). Resveratrol intracellular localization was confirmed by fluorescence confocal microscopy. Resveratrol nanoemulsion significantly improved the endothelial cell protection from H2O2-induced injury (750, 1000 and 1500 mu M H2O2) in comparison with incubation with the control resveratrol ethanolic solution (for example, 55 +/- 6% vs. 38 +/- 5% viability after 1500 mu M H2O2 challenge, p < 0.05). In conclusion, formulation of resveratrol as a SEDDS significantly improved its cellular uptake and potentiated its antioxidant properties on BAECs. (C) 2013 Elsevier B.V. All rights reserved.

An isolated mitochondrial complex III (CIII) defect constitutes a rare cause of mitochondrial disorder. Here we present the second case involving UQCRC2 gene, which encodes core protein 2, one of the 11 structural subunits of CIII. The patient has the same mutation (c.547C>T; p.Arg183Trp) as the first case and presented with neonatal lactic acidosis, hypoglycemia and severe episodes of liver failure. Our study expands the few reported cases of CIII deficiency of nuclear origin.

Sanfilippo disease (MPS IIIA) is an autosomal recessive lysosomal storage disorder resulting from sulfamidase deficiency, which is characterized by severe neurological impairment. Various tissues of MPS IIIA mice accumulate undegraded glycosaminoglycans and mimic the human neurodegenerative disorder, and are an excellent tool to both delineate disease pathogenesis and test potential therapies. The relationship between abnormal glycosaminoglycan storage and neurodysfunction remains ill defined. Pathways such as inflammation or oxidative stress have been highlighted in many neurodegenerative disorders, including lysosomal storage diseases, as major components of the neuropathology. By using quantitative polymerase chain reaction, we have compared the expression of selected genes in normal and MPS IIIA mouse cerebral tissues, focusing on inflammation, apoptosis and oxidative stress-related genes. We have identified several genes strongly over-expressed in the central nervous system of a MPS IIIA mouse, reflecting a neurological deterioration state. We have used these genes as markers to follow-up a long-term aspirin treatment. Aspirin treatment led to the normalization of inflammation- and oxidative stress-related mRNA levels in treated MPS IIIA mouse brains. A biochemical correction of an oxidative stress phenomenon both in the brain and peripheral organs of treated MPS IIIA mice was also obtained. These results suggest that anti-inflammatory intervention may be of potential benefit in MPS IIIA disease.

Glucose-induced oxidative stress is involved in endothelial dysfunction. Dimethylarginine dimethylaminohydrolase (DDAH) and arginase are regulators of the endothelial NO synthase (eNOS). This study aimed to compare the effect of two polyphenolic antioxidants, resveratrol and piceatannol, on DDAH and arginase pathways in bovine aortic endothelial cells under 25 mM glucose for 24 h. DDAH activity and expression were decreased in these cells as compared to control cells, whereas arginase activity was unchanged. DDAH inhibition led to intracellular accumulation of asymmetric dimethylarginine (ADMA), a natural inhibitor of eNOS. Under these conditions, cell pre-treatment with resveratrol (0.1-10 μM) restored basal DDAH activity and ADMA level with a dose-dependent effect. Piceatannol acted as resveratrol on DDAH pathway but at 10-fold lower concentrations. Resveratrol and piceatannol restored DDAH activity even in the presence of splitomicin, a specific inhibitor of Sirtuin 1. These results suggest potential therapeutic intervention targeting resveratrol or piceatannol administration to improve endothelial dysfunction.

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 micromol L(-1)) and linoleate (15-600 micromol L(-1)) below the critical micellar concentration (cmc=2000 micromol L(-1)), have been irradiated by gamma-rays (137Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min(-1)). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO*-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 micromol L(-1)) in the presence of BSA (15 micromol L(-1)), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO* radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 micromol L(-1)), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO* radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO* radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO*-induced linoleate oxidation.

Human immunodeficiency virus (HIV) infection is often complicated by the development of acquired immunodeficiency syndrome (AIDS) dementia complex (ADC). Implications of kynurenine pathway (KP) are suggested in ADC and other inflammatories brain diseases. The first and regulatory enzyme of the KP is the indoleamine-2,3-dioxygenase (IDO). IDO activation is known to contribute to the modulation of the immune response during various infectious diseases particularly in AIDS. HIV and viral proteins can activate IDO in immune cells leading to an increase catabolism of tryptophan through the KP; the consequence being the production of immuno-modulative and neuroactive metabolites. This mechanism is likely to favour HIV persistence. The present study analysed concomitantly several parameters involved in IDO regulation and activity associated with HIV-1-infection. We investigated relevant intracellular and extracellular mechanisms involved in the regulation of IDO expression and activity during the HIV infection and replication in human monocyte-derived macrophages (MdM). Using a complementary set of in vitro experiments, we found that HIV-1/Ba-L infection induces IDO expression and increases its activity in MdM. We also showed that IDO activation by HIV-1 is likely to be a direct effect of the infection and seems to be independent of IFN-gamma production.

Purpose of review Metabolic syndrome is very common and is associated with significantly increased risk for both cardiovascular disease and type 2 diabetes. At present, no unifying mechanism can explain it. However, insulin resistance is a key feature of this syndrome, plays a key role in triglyceride metabolism and contributes to dyslipidemia and development of type 2 diabetes. Here, we review the mechanisms involved in the overproduction of large VLDL and their catabolism and finally potential therapeutic targets to provide a more complete approach to treatment of these lipid abnormalities. Recent findings Dyslipidemia plays an important role in the development of atherosclerosis and is mainly associated by the hepatic overproduction of large triglyceride-rich VLDL, low levels of HDL cholesterol and high levels of small, dense, LDL cholesterol particles. It is thus of special interest to understand the mechanism involved in the hepatic synthesis of lipoproteins and the degradation of these lipoproteins that depend, to a large extent, on insulin action. Summary The atherogenic lipid abnormalities observed in the metabolic syndrome may require a combination of drugs such as statins and HDL-raising agents to provide a more complete approach to treating dyslipidemia and reducing cardiovascular risk.

The aim of this study was to investigate whether a relationship exists between the presence of low numbers of leukocytes in normal ovulatory cervical mucus and sperm quality and lipid content after migration. The percentages of live, motile and morphologically normal spermatozoa, movement parameters assessed by computer-aided sperm analysis (CASA), and ionophore-induced acrosome reaction measured by flow cytometry were determined before and after migration. High-performance liquid chromatography with ultraviolet detection was used to measure the sperm lipid content, including the various diacyl subspecies. The number of leukocytes found in solubilized mucus samples was counted using a haemocytometric method. Overall, the presence of leukocytes in the cervical mucus samples did not significantly influence sperm motility and morphology, sperm kinematic parameters, or the sperm content in sphingomyelin or cholesterol. In contrast, after migration, the decrease in various sperm diacyls and the level of induced acrosome reaction was significantly less pronounced in mucus samples containingor=10(4) leukocytes than in mucus samples with no or rare leukocytes whereas the level of induced acrosome reaction was higher. The present data suggest that the low level of leukocytes found in normal ovulatory cervical mucus could influence the process of sperm lipid remodelling/capacitation.

During pregnancy, the placenta ensures multiple functions, which are directly involved in the initiation, outcome of gestation and fetal growth. Human implantation involves a major invasion of the uterus wall and a complete remodeling of the uterine arteries by the extravillous cytotrophoblasts (EVCT) during the first trimester of pregnancy. Abnormality of these early steps of placental development leads to poor placentation, fetal growth defects and is very often associated with preeclampsia, a major and frequent complication of human pregnancy. Unexpectedly, genetic studies performed in mice established that the peroxisome proliferator-activated receptor-gamma (PPARgamma) is essential for placental development. In the human placenta, PPARgamma is specifically expressed in the villous cytotrophoblast (VCT) and the syncytiotrophoblast (ST) as well as in the EVCT along their invasive pathway. To study the mechanisms that control human trophoblast invasion during early placental development and to provide new insight in the understanding of preeclampsia, we have developed in vitro models of human invasive trophoblasts. We observed that activation of the ligand-activated nuclear receptor PPARgamma agonists inhibits the trophoblastic invasion process in a concentration-dependent manner. Analysis of PPARgamma-target genes revealed that placental growth hormone, the protease PAPP-A and the human chorionic gonadotropin hormone (hCG) might be involved in the PPARgamma-mediated effect in an autocrine manner. The presence of oxidized-LDLs at the maternofetal interface suggests that oxidized-LDLs from maternal sera might be a source of potential PPARgamma ligands for the trophoblasts. Indeed, oxidized-LDLs decrease trophoblast invasion in vitro and analysis of their content revealed that they contain potent PPARgamma agonists such as eicosanoids, but also oxysterols, which are ligands for another nuclear receptor, the liver X Receptor (LXR). LXRbeta was found to be expressed in trophoblast and LXR agonists shown to inhibit trophoblast invasion. Together, these data underscore a major role for PPARgamma in the control of human trophoblast invasion during early placental development and suggest that ligands such as oxidized-LDLs at the implantation site might contribute to the modulation of trophoblast invasion through activation of PPARgamma and LXRbeta, two nuclear receptors that modulate the human trophoblastic cell invasion process.

NADPH oxidase Nox2 is involved in the production of superoxide by rheumatoid synovial cells, constitutively and after pro-inflammatory cytokine treatment. The aims of the study were to evaluate the capacity of these cells to produce the superoxide anion in response to arachidonic acid (AA), and to study the involvement of cytosolic phospholipase A(2) (cPLA(2)) in the cytokine regulation of Nox2. Superoxide production was quantified in synovial cells obtained from six patients with rheumatoid arthritis (RA) and six with osteoarthritis (OA), stimulated with (i) AA, and (ii) PLA(2) inhibitors prior to IL-1beta or TNF-alpha treatment. Total cellular AA concentrations and PLA(2) activity were measured; effects of cytokines and NADPH oxidase inhibitors on the AA-activatable proton channel opening were also studied. Our results demonstrated that AA enhanced superoxide production in RA and OA cells; this production was significantly inhibited by iodonium diphenyl and apocynin. cPLA(2) inhibitors inhibited both IL-1beta and TNF-alpha-induced superoxide production in RA and OA cells. Basal PLA(2) activity was significantly more important in RA cells than in OA cells; PLA(2) activity was increased in IL-1beta and TNF-alpha pre-treated RA cells, and cPLA(2) inhibitors inhibited this activity. Opening of the AA-activatable proton channel was amplified when RA cells were pre-treated with both IL-1beta and TNF-alpha, and iodonium diphenyl and apocynin inhibited these cytokine effects. We concluded that AA is an important cofactor for synovial NADPH oxidase activity. Despite their direct effects on p47-phox phosphorylation, cytokines can also regulate the Nox2 activity though the AA-activatable associated H(+) channel.

This study investigated the effect of intranasal administration of progesterone on the early brain mitochondrial respiratory chain dysfunction and oxidative damage after transient middle cerebral occlusion in male and female mice. We showed that progesterone (8 mg/kg at 1 h post-middle cerebral occlusion) restored the mitochondrial reduced glutathione pool and the nicotinamide adenine dinucleotide-linked respiration in both sexes. Progesterone also reversed the decrease of the flavin adenine dinucleotide-linked respiration, which was only observed in females. Our findings point to a sex difference in stroke effects on the brain respiratory chain and suggest that the actions of progesterone on mitochondrial function may participate in its neuroprotective properties.

Oxidative stress plays a major role in the pathogenesis and in the onset of macrovascular complications of diabetes. We previously reported that the antihyperglycaemic drug metformin was able to decrease significantly intracellular reactive oxygen species (ROS) production of bovine aortic endothelial cells (BAEC) activated by high levels of glucose and angiotensin II (ANG). The aim of the present study was to investigate whether the antioxidant effect of metformin on BAEC could be mediated through a modulation of protein kinase C (PKC) activity, which plays a key role in the pathophysiology of diabetes. The effects of metformin on intracellular ROS production, PKC translocation and activity were studied on endothelial cells stimulated by PMA (a direct PKC activator), ANG or high levels of glucose as pathophysiological stimuli of endothelial dysfunction in diabetes. We showed that metformin decreased ROS production on PMA-, ANG- and glucose-stimulated BAEC in a similar manner to that obtained by PKC specific inhibitors (calphostin C, chelerythrine) alone. On the other hand, metformin reduced both PKC membrane translocation and kinase activity in ANG-stimulated cells. In PMA-activated cells, metformin reduced membrane PKC activity but we did not observe any alteration of PKC membrane translocation. Finally, in vitro incubation with purified PKC indicated that metformin had no direct effect on PKC activity. Taken together, our results suggest that metformin exerted intracellular antioxidant properties by decreasing ROS production through the inhibition of PKC activity.

This study aimed at investigating the in vitro protective effects of GWC22, a novel pinoline derivative [6-ethyl-1-(3-methoxyphenyl)-2-propyl-1,2,3,4-tetrahydro-beta-carboline] chlorhydrate, against radiation-induced oxidation of linoleate initiated by hydroxyl radicals ((*)OH). Using linoleate micelles (10(-2) m) as lipid model, two indexes of peroxidation have been measured, i.e. conjugated dienes and hydroperoxides. Similar determinations were performed with melatonin in order to compare the protective effects of the two compounds. It was observed that, the higher the concentration of GWC22 (or melatonin) (3 x 10(-5) to 10(-4) m), the stronger the antioxidant ability. In these in vitro assays, GWC22 showed a better antioxidant effect than melatonin for a given antioxidant concentration. A reaction scheme has been proposed to explain the inhibitory effect of an antioxidant via the propagating steps of the lipid peroxidation. Indeed, we have suggested that melatonin and GWC22 may compete with the fatty acid to scavenge lipid peroxyl radicals (LOO(*)). We have estimated a lower limit for the LOO(*) rate constant for GWC22 (>/=1.4 x 10(5)/m/s) and for melatonin (>/=2.8 x 10(4)/m/s) assuming that the k-value of the propagating step in linoleate (LOO(*) + linoleate) was 1.4 x 10(3)/m/s. The difference of reactivity between melatonin and GWC22 in this model system is assumed to be related to their relative lipophilicity.

Nutritional supplementation with glutamine, arginine and their precursors has been proposed to contribute to the protection against ischemia-reperfusion-related injuries. The aim of this study was to evaluate in an isolated perfused rat liver model the preventive effect of a 4-day oral ornithine alpha-ketoglutarate (OKG) supplementation against warm ischemia-reperfusion (I-R) injury, and the involvement of nitric oxide synthesis. Rats were fed a controlled regimen supplemented with either OKG (5 g kg(-1); n=15) or an isonitrogenous mixture of non-essential amino acids (Control; n=6) for 4 days. Livers were subsequently prepared for isolated perfusion experiments, including a 45 min no-flow ischemic period. The OKG-treated group was divided into two groups according to the absence (OKG; n=8) or presence of a NO-synthase inhibitor, L-N(omega)-nitro-arginine methyl ester (OKG L-NAME; n=7) during liver perfusion. Liver cytolysis after ischemia was demonstrated by an elevated alanine aminotransferase release during the last 15 min of reperfusion that was significantly higher in the OKG-L-NAME group. Tumor necrosis factor alpha (TNF(alpha)) production was transiently increased only in the control group just after ischemia. At the end of the reperfusion period, liver superoxide dismutase activity was significantly lower in the OKG-L-NAME group compared to control animals. Dietary OKG administration had only a limited effect in this model of mild hepatic I-R, leading mainly to reduced TNF(alpha) production. As the content of lipid peroxidation products was not modified, it seems that OKG acts on the inflammatory response rather than on oxidative reactions. This action can tentatively be attributed to the role of OKG as a glutamine precursor rather than to the synthesis of arginine and nitric oxide.

Beyond its antidiabetic activity justifying its use in the treatment of the type 2 diabetes, metformin (MET [dimethylguanidine, Glucophage]) has been shown to exhibit antioxidant properties in vitro, which could contribute to limit the deleterious vascular complications of diabetes. We investigated whether MET, at the pharmacological level of 10−5 mol/L, was able to modulate intracellular production of reactive oxygen species (ROS) both in quiescent bovine aortic endothelial cells (BAECs) and in BAECs stimulated by a short incubation with high levels of glucose (30 mmol/L, 2 hours) or angiotensin II (10−7 mol/L, 1 hour). Intracellular ROS production was measured by fluorescence of the DCF (2,7′-dichlorodihydrofluorescein) probe. Our results showed that MET was able to reduce the intracellular production of ROS in both nonstimulated BAECs (−20%, P < .05) and BAEC stimulated by high levels of glucose or angiotensin II (−28% and −72%, respectively, P < .01). Experiments performed in the presence of the nicotinamide adenine dinucleotide phosphate [NAD(P)H] oxidase inhibitor apocynin or the respiratory mitochondrial chain inhibitor rotenone indicated that MET exerted its effect partly through an inhibition of the formation of ROS produced mainly by NAD(P)H oxidase and also, to a lesser extent, by the respiratory mitochondrial chain.

Superoxide dismutases' (SODs) expression is altered in several diseases including Alzheimer, atherosclerosis, cancer and psoriasis. Previously, we reported a marked increase in Mn-SOD and Cu,Zn-SOD functional activity in human dermal psoriatic fibroblasts. As retinoic acid (RA) has been used in the treatment of psoriasis and a mechanism for its beneficial effects is not understood, we investigated the effects of RA on SOD mRNA and protein expression levels in human normal and psoriatic fibroblasts. Prior to RA exposure, Cu,Zn-SOD protein and mRNA levels were similar in normal compared to psoriatic fibroblasts while Mn-SOD protein and mRNA levels were increased in psoriatic cells. However, in contrast to normal fibroblasts, exposure of psoriatic fibroblasts to 1 microM RA down-regulated Mn-SOD mRNA, and also decreased Mn-SOD activity by approximately 80% with no change in Mn-SOD protein levels. In contrast, Cu,Zn-SOD protein and enzymatic activity were modestly reduced by RA treatment in both normal and psoriatic fibroblasts. Furthermore, RA treatment of psoriatic fibroblasts also caused a decrease in Cu,Zn-SOD steady-state mRNA levels. These results indicate that RA can serve as a regulatory agent to down-regulate the steady-state levels of both Mn-SOD and Cu,Zn-SOD in psoriatic cells. These findings offer a new model for the antiinflammatory activity of RA when used in the treatment of psoriasis.

Human embryonic implantation involves major invasion of the uterine wall and remodeling of the uterine arteries by extravillous cytotrophoblast cells (EVCT). Abnormalities in these early steps of placental development lead to poor placentation and fetal growth defects and are frequently associated with preeclampsia, a major complication of human pregnancy. We recently showed that oxidized low-density lipoproteins (oxLDLs) are present in situ in EVCT and inhibit cell invasion in a concentration-dependent manner. The aim of the present study was to better understand the mechanisms by which oxLDL modulate trophoblast invasion. We therefore investigated the presence of oxLDL receptors in our cell culture model of human invasive primary EVCT. We found using immunocytochemistry and immunoblotting that the lectin-like oxLDL receptor-1 was the scavenger receptor mainly expressed in EVCT and was probably involved in oxLDL uptake. We next examined the effect of low-density lipoprotein oxidative state on trophoblast invasion in vitro using EVCT cultured on Matrigel-coated Transwell. We demonstrated that only oxLDL containing a high proportion of oxysterols and phosphatidylcholine hydroperoxide derivatives that provide ligands for liver X receptor (LXR) and peroxisomal proliferator-activated receptor γ (PPARγ), respectively, reduced trophoblast invasion. We next investigated the presence and the role of these nuclear receptors and found that in addition to PPARγ, human invasive trophoblasts express LXRβ, and activation of these nuclear receptors by specific synthetic or natural ligands inhibited trophoblast invasion. Finally, using a PPARγ antagonist, we suggest that LXRβ, rather than PPARγ, is involved in oxLDL-mediated inhibition of human trophoblast invasion in vitro.

Metformin is an antihyperglycemic drug that exhibits some antioxidant properties. HO*-induced oxidation of metformin was studied in aqueous solution, in both aerated and deaerated conditions. Gamma radiolysis of water was used to generate HO* free radicals, capable of initiating one-electron oxidation of metformin. Oxidation end-products were identified by direct infusion mass spectrometry (MS) and high-performance liquid chromatography/mass spectrometry (HPLC/MSn): for every product, structure elucidation was based on its mass (simple mass spectra confirmed by HPLC/MS). In addition, fragmentation spectra (MS2, MS3 and MS4) and the determination of deuterium-hydrogen exchange sites provided valuable information allowing the complete identification of some of the end-products. At low radiation dose, four products were identified as primary ones, since they result from the direct attack of HO* radicals on metformin. These primary oxidation end-products were identified respectively as hydroperoxide of metformin, covalent dimer of metformin, methylbiguanide and 2-amino-4-imino-5-methyl-1,3,5-triazine. At high radiation dose, seven other products were identified as secondary ones, resulting from the HO*-induced oxidation of the primary end-products. A reaction scheme was postulated for the interpretation of the results.

Human implantation involves a major invasion of the uterine wall and remodeling of the uterine arteries by trophoblastic cells. Abnormalities in these early steps of placental development lead to poor placentation, fetal growth defects and are frequently associated with pre-eclampsia, a serious disease specific to human pregnancy. Lipid metabolism is altered during human pregnancy, with low-density lipoproteins (LDL) becoming more susceptible to oxidation. The aim of this study was to localize oxidized LDL (oxLDL) at the implantation site and to investigate the role of oxLDL in human trophoblast invasion in vitro. We showed by immunohistochemistry that oxLDL was present in cytotrophoblasts of villous and extravillous origin in sections of first trimester human placenta. We purified primary invasive extravillous cytotrophoblasts isolated from the chorionic villi of human first trimester placenta and cultured them on Matrigel-coated transwells. We demonstrated using this invasion assay that oxLDL, but not native LDL, inhibited cell invasion in a concentration-dependent manner. These results suggest that human trophoblast invasion may be modulated by oxLDL in vivo and provide new insights into the pathophysiology of pre-eclampsia associated with oxidative stress and defective trophoblast invasion.

Resume L'endothelium vasculaire joue un role primordial notamment dans la regulation du tonus vasculaire, en reponse a differents stimuli tels que la prostacycline, l’endotheline et surtout le monoxyde d'azote (NO • ). Le stress oxydant, caracterise par une augmentation relative des especes reactives de l'oxygene (ERO), peut diminuer la biodisponibilite du NO • , ce qui conduit a un dysfonctionnement vasculaire. En outre, les ERO (produites notamment par la NADPH oxydase) participent soit directement, soit par le biais des lipoproteines de basse densite oxydees, a la transduction du signal dans les cellules vasculaires. Elles sont ainsi impliquees dans les phenomenes d’apoptose, la proliferation des cellules musculaires lisses, l'adhesion des monocytes aux cellules endotheliales et l'agregation plaquettaire. Des perspectives therapeutiques visant par exemple a moduler la production radicalaire au niveau des cellules endotheliales pourraient etre envisagees pour restaurer la fonction endotheliale.